Replacement of α-galactosidase A in Fabry disease: effect on … · 2017. 4. 11. · effectiveness...

ORIGINAL ARTICLE

Replacement of α-galactosidase A in Fabry disease:effect on fibroblast cultures compared with biopsiedtissues of treated patients

Jana Keslová-Veselíková & Helena Hůlková &Robert Dobrovolný & Befekadu Asfaw &Helena Poupětová & Linda Berná & Jakub Sikora &Lubor Goláň & Jana Ledvinová & Milan Elleder

Received: 18 June 2007 /Revised: 21 December 2007 /Accepted: 20 January 2008 /Published online: 20 March 2008# Springer-Verlag 2008

Abstract The function and intracellular delivery of enzymetherapeutics for Fabry disease were studied in culturedfibroblasts and in the biopsied tissues of two male patientsto show diversity of affected cells in response to treatment.

In the mutant fibroblasts cultures, the final cellular level ofendocytosed recombinant α-galactosidases A (agalsidases,FabrazymeTM, and ReplagalTM) exceeded, by several fold,the amount in control fibroblasts and led to efficient directintra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, inthe samples from the heart and some other tissues biopsiedafter several months of enzyme replacement therapy (ERT)with FabrazymeTM, only the endothelial cells were free ofstorage. Persistent Gb3Cer storage was found in cardiocytes(accompanied by increase of lipopigment), smooth musclecells, fibroblasts, sweat glands, and skeletal muscle.Immunohistochemistry of cardiocytes demonstrated, forthe first time, the presence of a considerable amount ofthe active enzyme in intimate contact with the storagecompartment. Factors responsible for the limited ERTeffectiveness are discussed, namely post-mitotic status ofstorage cells preventing their replacement by enzymesupplied precursors, modification of the lysosomal systemby longstanding storage, and possible relative lack ofSap B. These observations support the strategy of earlytreatment for prevention of lysosomal storage.

Keywords α-Galactosidase A deficiency .

Enzyme replacement therapy . Persistent storage .

Enzyme targeting . Clearance of storage lysosomes

Introduction

Fabry disease (MIM 301500) is an X-linked deficiency ofα-galactosidase A (α-GALA, EC 3.2.1.22) leading towidespread lysosomal accumulation of glycosphingolipidswith terminal α-galactosyl moieties (mainly globotriaosyl-

Virchows Arch (2008) 452:651–665DOI 10.1007/s00428-008-0586-9

J. Keslová-Veselíková, H.Hůlková and R. Dobrovolný contributedequally to this work.

J. Keslová-Veselíková :H. Hůlková : R. Dobrovolný : B. Asfaw :H. Poupětová : L. Berná : J. Sikora : J. Ledvinová (*) :M. Elleder (*)Institute of Inherited Metabolic Disorders,Bldg. D, Division B; Ke Karlovu 2, 128 08,Prague 2, Czech Republice-mail: [email protected]

J. Keslová-Veselíkováe-mail: [email protected]

H. Hůlkováe-mail: [email protected]

R. Dobrovolnýe-mail: [email protected]

B. Asfawe-mail: [email protected]

H. Poupětováe-mail: [email protected]

L. Bernáe-mail: [email protected]

J. Sikorae-mail: [email protected]

L. GoláňClinical Department of Cardiology and Angiology,First Faculty of Medicine and University Hospital,U nemocnice 2, 128 08, Prague 2, Czech Republice-mail: [email protected]

e-mail: [email protected]

ceramide, Gb3Cer). The ensuing phenotype at the clinicallevel is well known, including its organ-limited mildervariants in hemizygous males and in females who aremosaic hemizygotes [9, 10]. The dominating therapeuticapproach, enzyme replacement therapy (ERT), is based onsubstitution of the missing enzyme with recombinant α-galactosidases, FabrazymeTM (agalsidase beta, Genzyme)or ReplagalTM (agalsidase alfa, SHGT) [6, 13]. Despitedifferences in the glycosylation pattern due to differentexpression systems [14, 41], both enzymes appear to befunctionally indistinguishable from each other [3, 23]. Theessential requirement for successful ERT of Fabry diseaseis the delivery of a sufficient dose of the enzyme into thestorage lysosomal compartment. The effect on storagehas been evaluated either by monitoring the level ofstorage or the induction of enzyme activity [12, 21, 3,23, 33]. However, there is not any report demonstratingcomplete clearance of the storage lysosomal systemgenerally. The effect is mostly restricted to endothelia,but the partial clearance effect has been seen by someauthors also in another cells, e.g., in the kidney [15].

The goal of this study was to compare effectiveness ofERT in two cell systems: by cultured mutant fibroblasts onone side, representing dynamic cell population minimallymodified by low degree spontaneous storage, and transientlyexposed to trace amount of tritiated substrate to follow itscatabolic pathway. This in vitro experimental system wascompared to the situation in vivo, represented by a series ofcell types of various regeneration capacities exposed toprolonged ERT after longstanding disease course. Extremecounterpart to cultured fibroblasts was represented bycardiocytes, postmitotic cells, modified by longstandingspontaneous storage.

This is also the first study focused on delivery of theapplied α-GALA into the storage cardiocytes which maycontribute to better understanding of the factors responsiblefor the limited effectiveness of ERT in vivo.

Patients

For details of examined patients, their ERT regime, and thebiopsy samples, see Table 1.

Materials and methods

The study was approved by the Ethics Committee of theGeneral Teaching Hospital and the First Faculty ofMedicine, Charles University (60/2003F), Prague. Cardiacbiopsy samples were taken during coronary surgeries.Written informed consent was obtained from patientsincluded in the study.

Cell lines Four fibroblast cell lines of patients with classictype of Fabry disease (mutations in the GLA gene:p.Q280K, p.R342Q, c.801ins36, c.delEx2 (c.195−?−369+?del)) came from the cell culture depository of the Instituteof Inherited Metabolic Diseases. Fibroblasts with mutationsc.801ins36 and p.Q280K were from patient 1 and patient 2,respectively, who underwent heart biopsy later on. All celllines (including p.R342Q and c.delEx2) were derived fromskin biopsies taken several years before ERT from patientswith classical form of the disease.

Recombinant α-galactosidases A REPLAGAL (agalsidasealpha, a gift from Shire Human Genetic Therapies, Cambridge,MA, USA) sample no. 001-0229 was used without any furthermodification (dilution 1mg/ml solution). FABRAZYME(agalsidase beta, a gift from Genzyme, Cambridge, MA,USA) batch no. H0214AM, diluted according to manufac-turer’s recommendation to 5mg/ml and then further diluted to1mg/ml with redistilled water.

Experiments in cell cultures Cultured skin fibroblasts weregrown in accordance with standard protocols in Dulbecco’smodified Eagle’s medium (DMEM) and 10% bovine fetalserum (BFS).

Preparation of substrate The globoside, Gb4Cer, wasisolated from human erythrocyte membranes, characterized,and tritium-labeled at the ceramide moiety (330MBq/mg).The (3H)Gb4Cer was incorporated into liposome particles(a mixture of phosphatidylcholine, α-tocopherol, andphosphatidic acid, Sigma, Czech Republic) and coated withapoprotein E [2].

Feeding assays DMEM, 3 ml per 25-cm2 flask, with 1%BFS and radiolabeled glycolipid (3.3 × 106dpm/1ml) wasapplied to nearly confluent Fabry fibroblasts and incubatedin a 5% CO2 incubator for 3days [2].

Uptake of enzyme Three milliliters of the enzyme solution(1.32μg of enzyme per milliliter, final activity 3.8μmolMU mg−1.h−1, the same for both enzymes) were placed infresh DMEM containing 10% heat-inactivated BFS andapplied to cells in 25-cm2 flasks. The flasks were thenincubated for different periods of time. At least two 25-cm2

flasks were analyzed for each time period/enzyme combi-nation (0, 12, 24, 48, 96, and 192 h).

Analysis of (3H)Gb3Cer degradation Three milliliters ofthe enzyme solution (0.66μg of enzyme per milliliter,final activity 1.9μmol MU mg−1 h−1, the same for bothenzymes) were placed in fresh DMEM containing 10%heat-inactivated BFS and conduritol B epoxide (finalconcentration 100mM, Calbiochem-Novabiochem GmbH,

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Germany) and applied to cells in 25-cm2 flasks preloadedwith (3H)Gb4Cer and incubated for different periods oftime. Analyses were performed at least in two 25-cm2

flasks for each time period/enzyme combination (0, 9, 14,19, and 24 h).

Cell harvesting and homogenization The fibroblasts wereharvested by trypsinization. The cell pellets were homog-enized in distilled water in a cuphorn sonifier filled with icefor 3 × 10s (Ultrasonic homogenizer 4710, Cole-Parmer,Chicago, IL, USA, 40% output). Protein concentration [16],enzyme activities, and uptake of the (3H)Gb4Cer tracerwere determined in homogenate aliquots.

Enzyme assay The activity of α-GALA was assayed inculture medium and in cell and tissue homogenatesaccording to the modified method of Mayes et al. [27].Briefly, the reaction mixture contained 5mM of substrate,4-methylumbelliferyl(MU)-α-D-galactopyranoside (Sigma,Germany), in citrate buffer (pH 4.5). For cell and tissuehomogenates, the reaction also contained 100mM α-N-acetyl-D-galactosamine to inhibit α-N-acetylgalactosaminidaseactivity. The complete reaction mixture was incubated for30min at 37°C, and the fluorescence of the released 4-MUwas measured at excitation 365nm and at emission 448nmon a luminescence spectrometer (Perkin Elmer LS50B,Wellesley, USA).

Extraction and thin-layer chromatography analysis of (3H)glycosphingolipids

The remaining cell homogenate was extracted withchloroform–methanol (2:1, v/v), and the lipids wereseparated on thin-layer chromatography (TLC) plates(Silica gel, Merck, Germany) using chloroform–methanol–water (70:30:05, v/v/v) as the solvent system. Chromato-grams were evaluated using a TLC linear radioactivityanalyzer (Raytest, Germany) [2].

Immunofluorescence labeling Fibroblasts were grown onglass coverslips to near-confluence using standard meth-ods. The cells were then grown for an additional 24h inDMEM containing 10% of heat-inactivated BFS and0.66μg/ml of the recombinant enzyme.

For the experiments using mannose-6-phosphate (M-6-P) receptor blocking, the M-6-P (Sigma, Germany) wasadded to the medium together with the enzymes to yield afinal concentration 10mM. Cells were fixed for 10min inice-cold methanol before immunodetection and non-specific binding was blocked with 5% BFS. Recombi-nant agalsidases were detected using rabbit polyclonalantibody against human α-GALA TK88 (anti α-GAL,TKT, Cambridge MA, USA) diluted to 9μg/ml, whilecathepsin D was detected simultaneously using mono-clonal mouse antibodies (Serotec, Oxford, UK) diluted to

Table 1 Patients’ data

Patient Age Phenotype Mutationin theGLA gene

α-GALA activityc innmol MU mg−1 proteinh−1 WBC skinfibroblasts

Biopsybefore ERT(year)

Biopsy in the courseof ERT: FabrazymeTM

(duration in months)

LV mass (g)before ERT

LV mass (g) inthe course of ERT(year of examination)

1 59a Classical c.801ins36 0.19(0.4%) 0.63(1.3%) Skin(1998)

LV (6) saphenous v.(21)heart auricle(21) biopsytaken 3 days after lastinfusion

440(2002Jul)

472 (2003Mar)817 (2004Jun)910 (2006Feb)840 (2007May)

2 50a Classical p.Q280K 0.13(0.3%) 0.95(1.9%) Skin(1980)

Heart auricle (65) skin (65)skeletal muscle (65) biopsytaken 2 days after lastinfusion

338(2001Jan)

404 (2002Jun)340 (2004Sept)423 (2006Jan)583 (2006Jun)521 (2006Dec)

3 60b Classical p.H46Y 0.14(0.3%) – – ND –

LV (left ventricle) mass was measured by echocardiography according to Sahn DJ et al., Circulation 58 (1978), pp 1072–1083.Clinical details and ERT regime: Patient 1 underwent two coronary bypass graft surgeries (April 2003, June 2004). Patient 2 underwent coronarybypass surgery in November 2006. Both patients were treated by Fabrazyme (dosage, 1 mg/kg every 2 weeks); patient 1 since October 2002 andpatient 2 since July 2002. Biopsy was taken on the third day after the protocolar infusion of Fabrazyme in patient 1 and on the second day after theinfusion in patient 2. Patient 3 underwent renal transplantation in 1993 and was treated by ERT (Replagal 0.2 mg/kg every 2 weeks) for 3 yearssince September 2001. ERT was withdrawn 1 year before death because of advanced dementia.ND not determineda Age when heart auricle biopsy was performedb Age at death. Only frozen autopsy samples of the heart were available for analysis which substituted approximate status without ERT.c Determined before ERT, control range for WBC 24.8–75.7 nmol MU mg−1 protein h−1 (n=65); for skin fibroblasts 33.8–70.8 nmol MU mg−1

protein h−1 (n=13)

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20 μg/ml. Gb3Cer was located using mouse monoclonalanti-Gb3 (CD77) antibody (BGR23, code 370680,Seikagaku, Japan) diluted to 6.7μg/ml after 10minfixation with 4% phosphate-buffered paraformaldehydeand blocking with 5% BFS. The primary antibodieswere visualized using Alexa Fluor 488-conjugated goatanti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes, Invitrogen, Carlsbad, CA,USA). The samples were examined using two Nikonconfocal microscope setups (1) Nikon C1, Eclipse E800microscope equipped with a laser scanning confocalhead (Nikon, Japan) and a Nikon 60X, NA 1.4 oilimmersion objective or (2 Nikon C1si LSM headmounted on TE-2000 inverted microscope and withNikon TIRF NA 1.49 60× oil immersion objective. Allchannels were recorded separately to avoid cross-talkbetween channels. The co-localization analysis wasperformed with representative images using ImageJsoftware [31] with JaCoP plugin [4] and Huygens imageanalysis software (SVI, Hilversum, The Netherlands). Thedegree of co-localization was assessed by coefficientsdescribed by Manders et al. [26]. Two independentexperiments were done to support the results.

The image acquisition conditions were kept constant inall parameters for samples presented in Fig. 3e–h.

Histochemical and ultrastructural analyses of tissue sam-ples Patient 1. The sample of superficial part of the leftcardiac ventricle biopsied after 6months of ERT wasprocessed for electron microscopy. Specimens of the heartauricle and part of the saphenous vein graft taken after21months of ERT were transferred to the liquid nitrogenand stored on dry ice.

Patient 2. Samples of the heart auricle, skin, and skeletalmuscle taken after 65months of ERT were frozen in liquidnitrogen or fixed with phosphate-buffered paraformalde-hyde (Fluka, Buchs, Switzerland). Samples for standardultrastructural analysis were fixed with 10% paraformalde-hyde overnight, post-fixed in phosphate-buffered 1%Os04,dehydrated and embedded into Epon-Araldit mixture.Samples for electron microscopic immunostaining werefixed with 4% paraformaldehyde for 2 h, dehydrated, andembedded into LR White resin (London Resin Company,Reading, England). Skin biopsies taken before ERT startfrom both patients were processed for standard electronmicroscopy. Patient 3 (death one year after ERT termina-tion). Postmortem frozen and resin embedded samples fromthe left cardiac ventricle were included into the study tosubstitute approximate status before ERT.

Cryostat sections were prepared from the frozen samplesand used for immunohistochemical evaluation of thelysosomal storage status (Gb3Cer storage, lipopigment

accumulation) for detection of the applied agalsidase proteinand for histochemical evaluation of its activity.

Lysosomal storage status Gb3Cer storage was detected incryostat sections after pretreatment with cold anhydrousacetone (at 4°C) for removal of possible apolar lipidadmixture and for glycolipid fixation. They were brieflypost-fixed with 4% buffered paraformaldehyde (for 5min at4°C). After standard blockade of endogenous peroxidase(with sodium azide and H2O2) and non-specific protein–protein interactions (with 5% BFS), the sections wereincubated with monoclonal mouse anti-Gb3Cer antibodydiluted 1:100 in 5% BFS (at 37°C for 1h or at 4°Covernight). Detection of bound primary antibody wasachieved using EnVision + System-HRP Anti-Mouse(DakoCytomation, Glostrup, Denmark) and the DAB +Chromogen kit (K 3460, DAKO Cytomation, Carpinteria,USA). The staining specificity was checked using total lipidextraction by chloroform (C)–methanol (M)–water (W)4:8:3, followed by C–M 2:1 (each step for 30min at roomtemperature). The sections were rinsed in methanol, brieflyfixed in paraformaldehyde, and used for the lipid immuno-detection. Lysosomal storage status was also studied bybirefringence of liquid crystals of the stored polar lipid andby detection of lipopigment autofluorescence. The latterwas evaluated by epifluorescence microscopy using theNikon BV-2A filters blocks (EX 400-440, DM 455, BA470). The best results were obtained after brief (5min) pre-extraction of the lipids with C–M mixture (1:1, v/v) toprevent moderate non-specific autofluorescence of thestored lipid. The liquid crystalline state of the stored lipiddeposits exhibited considerable instability and was bestevaluated using Baker’s fixative solution. The results werecompared with samples from age-matched controls. Neutralterm lipopigment was used throughout the manuscriptbecause of ambiguous terminology [34].

Immunohistochemical detection of the applied enzymeprotein Cryostat sections were fixed with 4% phosphate-buffered paraformaldehyde exposed to standard blockingprocedures (see above) and incubated for 1h at 37°C withthe primary polyclonal antibody TK 88 diluted to 4.5μg/mlin 5% FCS then washed in PBS and exposed to thedetection kit (EnVision + System-HRP Anti-Rabbit, Dako-Cytomation). An alternative fixation of sections using amixture of chloroform–acetone (1:1, v/v) for 30min at roomtemperature followed by paraformaldehyde was used forsome of the experiments. Semi-thin 0.5-μm sectionsembedded into LR White medium were treated with citratebuffer pH6.0 for 40min at 90°C and incubated after theaforementioned blocking procedures with TK88 antibodyfor 1h at 37°C and thereafter treated with the detection kitas described above.

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The sections immunostained either for Gb3Cer or forα-GALA were counterstained with Harris hematoxylinand mounted into the Immu-Mount medium (Shandon,Pittsburgh, PA, USA) which allowed simultaneous evalu-ation of the presence of the applied enzyme (eventuallyGb3Cer), birefringent lipid and autofluorescent lipopigment.

Histochemical detection of in situ enzyme activities Activ-ity of α-GALA was evaluated using both the post-couplingtechnique with 6-bromo-2-naphthyl-α-D-galactopyranoside(LaChema, Brno, Czech Republic) and Fast Blue B andthe simultaneous azo-coupling technique with 1-naphthyl-α-D-galactopyranoside (Koch Light, Colnbrook Bucks,UK) and hexazonium pararosaniline; both were done onsemi-permeable membranes allowing to detect activities ofboth bound and soluble enzymes according to the publishedprescriptions [24]. Semi-permeable membrane techniquewas used for demonstration of activities of β-galactosidase(indigogenic method), β-glucuronidase, and acid phospha-tase (simultaneous azo-coupling method using in both casesnaphthol ASBI-type substrates and hexazonium pararosan-iline). Acid phosphatase activity was also demonstratedusing the aqueous medium [24].

Mannoso-6-phosphate receptor (MPR, cation-independent)was detected using monoclonal antibody (2G11 clone,Abcam, Cambridge, UK), diluted 1:400. Polyclonal antibodyagainst cathepsin D was purchased from DAKO and used indilution 1:200. In both cases, the incubation was overnight at4°C.

Results

Studies in cultured mutant cells

Uptake of recombinant enzymes into cultured Fabry cellsand efficiency of Gb3Cer hydrolysis

To monitor the function of agalsidases in situ, the uptakeinto Fabry fibroblasts was measured over the course of192 h (8days). Both enzymes were similarly internalizedinto the cultured cells, with maximum activity at 24hincubation. The uptake of agalsidase beta measured withMU-substrate was 2.5-fold greater than that of agalsidasealpha, although the enzymes were applied at the sameconcentration (1.32mg/ml medium). This result was con-firmed by repeated experiments on three different Fabryfibroblast cell lines. After 24h, the cellular activities of bothenzymes started to decline. However, even after 8days, thevalues exceeded several times the normal activity of healthycells (Fig. 1). Activity in the culture medium decreasedequally in both agalsidases, falling to 4–6% of its originalvalue (Fig. 1, inner graph).

The correction of the defect in Fabry cells was monitoredwith natural substrate in mutant cell cultures pre-loaded withtritium-labeled globoside (3H)Gb4Cer, the (

3H)Gb3Cer pre-cursor. The quantity of degradation products calculated fromTLC-radioactivity scans reflected approximately the uptaketendencies of the respective enzymes (Fig. 2). Despite thedifferences in uptake (Fig. 1), both loaded agalsidases provedefficient in intra-lysosomal degradation of Gb3Cer.

Confocal microscopic examination of internalizedagalsidases with endosomal/lysosomal markers in culturedFabry cells

Fabry fibroblasts were incubated with recombinant agalsidasesfor 24h. The intracellular distribution of agalsidase alpha andagalsidase beta in the cells was detected by anti α-GALAantibody (red channel, Fig. 3a,b). The lysosomes werevisualized in the perinuclear region by immunodetection ofcathepsin D (green channel, Fig. 3a,b). In a similar manner,the localization of intracellular Gb3Cer deposits wascorrelated with recombinant enzymes internalized into α-GALA deficient fibroblasts (Fig. 3g,h). The merger of theagalsidase and cathepsin D signals suggested lysosomallocalization of loaded recombinant enzymes and was furtherdocumented by co-localization maps (Fig. 3c,d). Correlationof agalsidase signal with lysosomal transmembrane proteinLAMP 2 was also demonstrated (data not shown).

Endogenous enzyme protein was not detected in the cellsof Fabry patients (data not shown). The addition of M-6-Pto the culture medium, together with agalsidases, strongly

Fig. 1 Internalization of recombinant α-galactosidases A (agalsidasesalpha and beta) into Fabry skin fibroblasts as a function of time.Recombinant enzymes (1.32 μg of enzyme protein per milliliter ofmedium) were applied into cultures of Fabry cells incubated understandard conditions. After 0, 12, 24, 48, 96, and 192 h, medium wasremoved, cells harvested, and analyzed in vitro with MU-substrate asdescribed in “Materials and methods”. Each value is average ofduplicate flasks. Intracellular agalsidase beta activity exceeded 20times normal levels at the maximum (24 h), while agalsidase alphaactivity was eight times the normal value in healthy cells (50.3±13.6 nmol MU mg−1 protein h−1 in controls, see broken line). Insert isa time course of agalsidases stability in the culture medium

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inhibited the uptake of both enzymes (Fig. 3e,f), confirmingthat M-6-P receptor endocytosis is a major pathway ofagalsidase transport into fibroblasts. The double labeling ofGb3Cer (Fig. 3g,h, green channel) and recombinantenzymes (Fig. 3g,h, red channel) did show only a fewgranular structures containing non-degraded Gb3Cer whichwere still present. The co-localization map of Gb3Cer andrecombinant enzymes (Fig. 3i,j) showed only limitedoverlap (compare logarithmically color coded overlapcoefficient in co-localization maps of Gb3Cer/α-GALAand CatD/α-GALA).

Studies in tissues

In vitro α-GALA activity in the heart samples

α-GALA activity determined in the auricular sample of thepatient 1 was 1.53 vs. 10.18±2.32 nmol MU mg−1 proteinh−1 in controls (n=5). This value represented 15% of thenormal activity of the enzyme in the auricular part of theheart which implies slight increase from deficient values.There was no representative sample of the heart auricle inpatient 2 for biochemical analysis. Residual enzymeactivity in the autopsy sample (patient 3) was barelydetectable (0.12 nmol MU mg−1 protein h−1, i.e., about1% of the control mean).

Immunohistochemical and ultrastructural studies

Evaluation of the lysosomal storage status Before ERT,dermal capillaries (skin biopsy available in patients 1 and 2)displayed lysosomal storage (Fig. 4a). ERT led to completeclearance of the capillary endothelium lysosomes alreadyafter 6 months (patient 1) from the stored lipid and toretraction of the originally expanded system to normaldimensions (Fig. 4b,c). Capillary basement membraneswere thin and single. Capillary endothelial storage invarious degrees was detectable in the postmortem heartspecimen of the patient who died 1 year after ERTwithdrawal (Fig. 4d).

The auricular cardiocytes in both patients on ERTdisplayed variable, but on average high degree, Gb3Cerstorage concentrated in the large perinuclear region(Fig. 5a). Lipid storage was paralleled by increase inlipopigment (Fig. 5c). Cardiocytes free of lysosomal lipidstorage and containing only lipopigment were rare, andthose free of both lipid and lipopigment were exceptional.The postmortem heart specimen displayed massive anduniform lipid storage occupying the bulk of the cardiocytecytoplasm (Fig. 5b). Lipopigment was present in the formof dispersed tiny granules (Fig. 5d). Ultrastructure ofcardiocytes, studied in all three cases, showed numerouslysosomes with varying degrees of aggregation and fusionwith deposits of loosely or densely packed lipid mem-

Fig. 2 Degradation of (3H)Gb3Cer by Fabry fibroblasts loaded with(3H)-globoside after application of agalsidases alpha and beta.Illustration of 24-h experiments (example of one experimental setfrom three is shown). (3H)-globoside (5×106 dpm), precursor of (3H)Gb3Cer, was incorporated into the liposomes coated with Apo E andadded to the culture medium as described in “Materials and methods”.After 3 days of incubation, the agalsidases were applied at finalactivity of 1.9 μmol ml−1 h−1. After 0, 9, 14, 19, and 24 h, cells wereharvested, the lipids extracted, separated by high performance thinlayer liquid chromatography, and the products were quantified by TLClinear radioactivity analyzer (“Materials and methods”). Percentage ofdegradation products was calculated from TLC scans in relation tototal cell radioactivity comprising substrate (3H)Gb3Cer and the sumof its degradation products partially transformed into products of re-synthesis (phospholipids)

Fig. 3 Co-localization of internalized agalsidase alpha (a–i) andagalsidase beta (b–j) with cathepsin D and Gb3Cer in the Fabryfibroblasts after 24-h incubation (a, b) Confocal analysis of cellslabeled for α-galactosidase A (α-GALA, red) and intra-lysosomalmarker cathepsin D (CatD, green) displayed granular distribution ofboth signals and their parallel considerable overlap. Mander’scoefficients MGAL and MCatD ranged from 0.209 to 0.441 and 0.53to 0.849 in the images, respectively. MGAL and MCatD coefficientsrepresent the fraction of co-localizing pixels from the overall numberof pixels with intensity values above the given threshold calculated forthe particular image channel. Threshold levels to filter backgroundwere set to 10% of the pixel intensity range in both channels. The co-localization of both signals is also illustrated by co-localization maps(c, d) employing overlap coefficient values (0–1), color coded bylookup table placed under this panel. Addition of 5 mM of M6P to theculture medium completely blocked the α-GALA transport to thelysosomes (e, f) and changed the signal of α-GALA from granular(intra-lysosomal) pattern to diffuse low-intensity signal virtuallyinseparable from nonspecific background. The co-labeling of α-GALA and Gb3Cer in the cultured fibroblasts of Fabry patientsincubated 24 h with recombinant agalsidases showed very limitedoverlap (g, h). The distribution of co-localization of both signals isalso demonstrated by co-localization map of overlap coefficient (i, j).Comparing images (g, h) and (i, j) demonstrates minimal correlationof α-GALA and Gb3Cer signals in the cells. This observationsuggests that the population of lysosomes with strong α-GALAdetection has already been cleared off Gb3Cer. Scale bars in f (validfor e, f) and j (valid for a–d and g–j) correspond to 10 μm

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branes often mixed with pleiomorphic lipopigment depos-its (Fig. 6a). In patient 2, there were exceptionalcardiocytes with discrete lysosomal lipid storage but withnumerous small lucent vesicles, suggesting relativelyeffective lysosomal clearance (Fig. 6b).

In all cases, irrespective of the ERT, the vascular smoothmuscle cells displayed marked lipid storage. Persistentstorage was seen in the skin eccrine glands (Fig. 7a) inpericytes and stromal fibroblasts, especially those in thehair bulb matrix (Fig. 7b). Discrete storage was found in theskeletal muscle fibers (not shown here). In the sample ofthe saphenous vein graft, there was prominent lipid storagein intimal cells and in the smooth muscle cells of the tunicamedia (Fig. 7c), which also displayed hypertrophy andhyperplasia. Endothelial cells of the tunica intima were freeof storage, as were the endothelial cells of the adventitialvessels.

Storage in some cell types was accompanied by increasein lipopigment (mostly in the cardiocytes, skeletal muscle,but almost absent in the smooth muscle cells). Electronmicroscopy showed densely packed lipid membranes andpleiomorphic structure of the lipopigment in lysosomes.

Evaluation of the intracellular delivery of the appliedenzyme In situ immunodetection of the applied enzymeperformed in heart samples of the ERT-treated patientsshowed relatively strong signal of variable intensity in amajority of cardiocytes (Fig. 8a). Simultaneous detection ofboth the immunostained agalsidase protein and the storedbirefringent lipid/lipopigment in a single section showedthe coexistence of both the targeted enzyme and lipid

storage in a very close contact in the majority of cardiocytes(Fig. 8c). More intimate contact between the targetedenzyme and the storage compartment was seen after briefextraction of the stored lipid with chloroform–acetonemixture (see “Materials and methods”; Fig. 8d). Unfortu-nately, the applied enzyme proved to be fixation sensitive,so that our attempts to define its relation to the storagecompartment at the ultrastructural level failed. However, insemi-thin sections exposed to intensive antigen retrieval,there were numerous positive granules of the appliedenzyme interspersed between autofluorescent lipopigmentgranules (Figs. 8e,f). Both enzyme histochemical techni-ques (see “Materials and methods”) showed the presence ofactive enzyme in the storing cardiocytes (Fig. 9a). Therewas also a close relation of the enzyme product to thestorage lysosomal compartment better seen in simultaneousazo-coupling technique. Strong immunohistochemicalsignal for the enzyme protein was seen in the vascularand endocardial endothelium and in the epicardial meso-thelium in both patients on ERT. Neither the enzymeprotein nor its activity was detectable in cardiocytes in theFabry postmortem heart sample without ERT (patient 3;Figs. 8b, 9b).

Other lysosomal enzymes in the cardiocytes The storageatrial cardiocytes displayed strong and uniform induction ofacid phosphatase activity (Fig. 10a). In contrast, inductionof β-galactosidase activity was highly irregular (Fig. 10b).Activity of β-glucuronidase was barely detectable. Activ-ities of the above enzymes in autopsy samples wereuniformly low.

Fig. 4 Ultrastructure of the storage in the heart capillary endothelium.a Capillary endothelium with prominent storage before ERT. Patient 2(bar 2 μm). b Capillary endothelium free of storage after 65 months ofERT. Persisting storage in pericapillary cells. Patient 2 (bar 4 μm). cDetail of the capillary endothelium after 6 months of ERT with

discrete lysosomal system containing unspecific lipopigment. Patient 1(bar 2 μm). d Lysosomal storage in the capillary endothelium of theheart after 1 year of ERT termination. This picture represents extremein the wide range of capillary storage levels. Patient 3 (bar 2 μm)

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Signals of both MPR (Fig. 10c) and cathepsin D (notshown) in the biopsy samples were remarkably heteroge-neous in storage cardiocytes ranging from barely detectableto very strong intensities.

Discussion

Studies in cell culture Higher uptake of beta- than alpha-agalsidase reflected greater number of phosphorylated

Fig. 6 Ultrastructure of the car-diocyte storage. a Patient 2.Atrial cardiocytes after65 months of ERT with numer-ous individual lysosomes accu-mulating lipid dominating overlipopigment (bar 2 μm). b Ex-ceptional finding in the samepatient showing only discretelipid storage lysosomes. Thereare numerous small vesicleswith lucent content, which mostprobably represent cleared lyso-somes (bar 2 μm)

Fig. 5 Persisting lipid storage in cardiocytes. a, b Immunohistochem-ical demonstration of Gb3Cer. a Cardiocytes of the heart auricle(patient 2) after 65 months of ERT (bar 100 μm). b Cardiocytes of theleft ventricle. Autopsy (patient 3) 1 year after ERT cessation. Insertshows absence of Gb3Cer staining in control cardiocytes (bars100 μm). Note certain degree of variability of storage, reflectingparallel excessive lipopigment deposition. c, d Increase of the

autofluorescent lipopigment demonstrated after brief lipid extractionwith C–A. c Large lipopigment clusters in heart auricle cardiocytes(patient 2). Insert: age-matched control (bars 100 μm). d Cardiocytesin the left ventricle (patient 3) with dispersed numerous discretelipopigment granules (bar 100 μm). Insert: age-matched controlshowing coarser lipopigment granules restricted to the perinuclearregion (bar 50 μm)

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mannose moieties per beta enzyme molecule [23, 33] andpresumed principal role of MPR-mediated endocytosis [18]in skin fibroblasts which are receptor-rich. The completeinhibition of cellular uptake of both agalsidases by M-6-P isin accord with this conception.

Lysosomal targeting of the applied active enzyme wasdouble-checked. Firstly, successful targeting of enzymetherapeutics to lysosomes has been proven through co-localization with lysosomal marker cathepsin D. Evaluationof images based on calculation of correlation and co-localization coefficients pointed to slightly lower co-localization parameters for the alpha compared to the betaenzyme.

It must be pointed out that enzyme endocytosis andtrafficking via early to late endosomes is a dynamicprocess. Therefore, only partial co-localization withlysosomal markers can be achieved under the conditionof continuous enzyme supply. Final evaluation ofGb3Cer and α-GALA signals in Fig. 3i,j stronglysuggested that lysosomes in fibroblast population werealmost cleared of substrate after 24-h incubation with anyof the enzymes.

Secondly, the delivered enzyme was shown to degradeefficiently the natural substrate (3H)Gb3Cer produceddirectly in lysosomes from its radiolabeled precursor (3H)Gb4Cer. The exact quantification of lysosomal (

3H)Gb3Cerdegradation in situ confirmed indirect observation thatrecombinant agalsidases degrade accumulated Gb3Cer ina dose-dependent manner [33].

In situ (3H)Gb3Cer degradation revealed lower differ-ences between the two enzymes than in vitro assay withsoluble MU-analog using the whole homogenate ofagalsidase-loaded fibroblasts. This may be related to theintracellular transport and unspecified location of agalsidasein in vitro assay and to the availability of Sap B inlysosomes when supply of enzyme is high.

However, despite the different degree of agalsidaseendocytosis, the cell-associated activity of both enzymesexceeded by several times the α-GALA activity of normalhealthy cells (see Fig. 1), and both were found effective incleavage of (3H)Gb3Cer. Their stability in the medium wasfound identical in accordance with findings in blood plasma[33].

Our experiments demonstrated effective targeting of theapplied enzyme to fibroblast lysosomes using both co-localization and cleavage of its substrate locally producedfrom its exogenously added precursor. (3H)Gb4Cer wasused for the first time as an intra-lysosomal tracer offunction of recombinant agalsidases that does not increasethe substrate load and can serve as a model system forevaluation of catabolic lysosomal functions and theirdefects.

To sum up, the ERT is effective in the cultured fibroblastsystem. In our view, it is because Fabry fibroblastsrepresent a short-lived dynamic cell population unmodifiedby low degree of spontaneous storage. Such cells exposedtransiently to a trace amount of labeled substrate caneffectively endocytose the recombinant enzyme. The

Fig. 7 Persisting storage in other cell types. a, b Patient 2 (ERT65 months). Birefringent stored lipid in eccrine skin glands (a) (bar100 μm) and in hair follicle matrix fibroblasts (b) (bar 50 μm). c

Patient 1 (21 months on ERT). Graft of saphenous vein used forcoronary artery reconstruction with massive birefringent deposits ofthe stored lipid (bar 100 μm)

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Fig. 8 Immunohistochemical detection (TK 88 antibody) of α-GALAprotein in cardiocytes. a Strong signal for the enzyme protein in atrialcardiocytes in patient 1 (21 months on ERT; bar 100 μm). b Absenceof signal in ventricular cardiocytes in patient 3 (1 year after ERTwithdrawal). Insert: signal for endogenous α-GALA in controlventricular cardiocytes (bars 100 μm). c Simultaneous immunohisto-chemical detection of the enzyme and of the birefringent stored lipidshowing coexistence of both in same cells. Semi-crossed Nicols (bar

30 μm). d Immunohistochemical detection of the enzyme after brieflipid pre-extraction shows close relation of the enzyme to the storagecompartment (bar 50 μm). e Immunohistochemical detection of theenzyme in a semi-thin section of LR-White-embedded specimen (bar20 μm). f The stained section was simultaneously examined under UVto show lipopigment (unstained) which represents marker of thestorage compartment (bar 20 μm). c, d Patient 1; e, f patient 2

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enzyme is delivered to the storage compartment where itexerts its degradative function. The whole process isfacilitated by easy access to the applied enzyme via MPRand by the possibility to endocytose it along the entire cellperimeter of unpolarized cell.

Studies in tissues Our study showed effective clearing ofendothelial cells by ERT corresponding to the publishedresults [21, 7, 36]. The design of our study allows us toconclude that endothelial storage is resumed within 1 yearafter cessation of ERT. In the Fabry disease mouse model,the storage was resumed after several weeks of withdrawalof the enzyme [21].

In contrast to endothelia, the storage persisted in a set ofcells despite the continuous ERT, e.g., in cardiocytes,vascular smooth muscle cells, pericytes, some fibroblasts,and perineurial cells, all repeatedly described to resistsubstantial storage reduction by ERT [12, 40, 7, 39, 43, 32,33, 36]. We may add skin eccrine glands, hair bulbfibroblasts, and skeletal muscle. The design of the studydid not allow detecting possible diminution of the persistingstorage mentioned in some of the above-quoted reports. Forinstance, the cardiocyte storage pattern in both our casesadmits partial clearance (Fig. 6b). Persistent storage incardiocytes was accompanied by a notable increase inlipopigment which should be taken as irreversible. Thesefindings may explain the absence of cardiac improvementin either patient on ERT (see Table 1).

It is not easy to explain the limited ERT effectiveness.There is the undisputed role of the physical barriers,represented by fibrous stroma and basal membranes,especially when multiplied around capillaries [11]. Freeaccess of the applied enzyme to endocytotically active cellperimeter in non-polarized cultured fibroblasts and through

fenestration in the liver sinusoidal endothelium to thehepatocyte basolateral pole may contribute significantly tothe effective ERT [35] (also M.E. personal observation),similarly as the number of MPR known to be of highdensity in fibroblasts [42, 18]. However, the density of thelatter was not shown to correlate with the level of theapplied enzyme uptake [29]. In polarized storage cells(epithelia, e.g., in the sweat glands, respiratory epithelium,etc.), the applied enzyme uptake is restricted to thebasolateral pole. In case of renal tubules, both poles mayparticipate to different degree in various parts of thenephron [38]. In the case of endothelial cells, thereproducible effectiveness of ERT may be explained bythe barrierless access of the enzyme to the apical pole activein endocytosis [37].

Our findings point to further potentially important facts.Before all, it is the presence of the applied active enzymeinside the storage cardiocytes in patients under ERT (not insamples from patient 3 with ERT terminated). We base thiscrucial finding on the following data. No enzyme proteinwas immunohistochemically detectable in cultured mutantfibroblasts of both patients. As the value of enzyme activityin wild-type fibroblasts is much higher (approximatelyfivefold) than in the heart, it excludes presence ofdetectable amount of endogenous mutant enzyme in atrialcardiocytes of the Fabry patients studied. This is in accordwith negative results in the heart from patient 3 with ERTterminated (see Fig. 8b). The histochemically demonstrableα-GALA activity in cardiocytes of ERT-treated patients(and not in patient 3) fits perfectly with the conclusion(Fig. 9). Similarly, increase in activity was shown bybiochemical assay in one auricular sample available. Wewould also like to stress that in our studies, we usedcryostat sections because the enzyme was found to be

Fig. 9 Histochemical evaluation of α-GALA activity with simulta-neous azo-coupling method. a Presence of enzyme activity in storingatrial cardiocytes of patient 1 (21 months on ERT; bar 100 μm).

Compare with Fig. 8. b Absence of enzyme activity in ventricularcardiocytes 1 year after cessation of ERT (patient 3). Insert showsenzyme activity in control ventricular cardiocyte (bars 50 μm)

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remarkably fixation-sensitive (see below). Therefore, sen-sitivity of our detection of the applied enzyme was higherthan that in previous reports [20, 29].

The sensitivity of the applied enzyme to aldehydefixation prevented its localization inside storage lysosomesat the electron microscope level. However, results in bothcryostat and LR-White-embedded sections (Fig. 8a,d–f)indicate at least partial successive delivery of the enzyme.Its quantity may play a role, as the heart in Fabry diseasemay express storage even in cases with relatively highresidual activity [30]. We would like to hypothesize thatthere may be modification of membranes of the endosomal/lysosomal compartment, induced by longstanding storage,especially in cells with low regeneration potential, cardio-cytes being an ideal example. Such modification could leadto decreased effectiveness of the contact of the storagecompartment with the endocytotic delivery of the appliedrecombinant enzyme. Decrease in MPR in many of thestorage cardiocytes suggests a changed proportion oflysosomes to late endosomes, and this may result in alteredfusion, inevitable for delivery of the applied enzyme cargoto the latter [8, 25]. It might also indicate disturbed contacteven with the Golgi apparatus, which might explain lowactivities of β-glucuronidase, β-galactosidase, and cathep-sin D in the cardiocyte storage compartment. Further,parallel deposition of lipopigment (see above) points toirreversible modification of storage lysosomes. Any rele-vant comparison with the mouse model of Fabry disease isdifficult, as the mouse cardiocytes do not seem to displaystorage regularly [21]. Nothing is known about the effectiveinteraction of the applied agalsidase with Sap B in Fabrydisease. The disorder, featured by densely packed lyso-somal lipid membranes, differs substantially from the well-defined physiologic conditions [22]. Decreased amount ofSap B in Fabry disease [28] may aggravate the situation. Insum, one of the critical factors in ERT of Fabry diseasemay, thus, be the degree of storage and physical status ofthe stored Gb3Cer and availability of Sap B. All this raisesan important question whether the effective targeting of theenzyme to the storage cell is the only prerequisite foreffective ERT.

The last factor awaiting evaluation is the role of storagecell turnover in Fabry disease (and in other lysosomalstorage disease). In the case of endothelial cells in Fabrydisease, preliminary results (J. Bultas, personal communi-cation) showed its increase. Therefore, the positive effect ofERT might be also explained as a replacement effect ofstorage cells by the endothelial cell progenitors [1, 5, 17]that may easily endocytose the applied enzyme and thusprevent storage. This mechanism does not come intoconsideration in the case of post-mitotic storage cells,typically cardiocytes. Taken together, our observationsspeak in favor of the strategy of early treatment to prevent

Fig. 10 Lysosomal enzyme activities and MPR immunostaining instorage atrial cardiocytes. a High and uniform activity of acidphosphatase (aqueous medium). Insert shows activity in controls(bars 50 mm). b Heterogenous activity of β-galactosidase withremarkably low values in some cardiocytes. Insert shows activity incontrol cardiocytes (bars 50 mm). c Similarly heterogeneous signal forMPR (bar 30 mm)

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development of storage and of irreversible modulation ofpost-mitotic cells or of cells with low regeneration potential(cardiocytes, smooth muscle cells) and of their lysosomes.This would be the most effective prevention of irreversibletissue changes. Studies on mouse model which wouldcompare effectiveness of early ERT (starting immediatelyafter birth) with its delayed application in adulthood aredesirable.

Conclusions

We suggest that future studies should focus on defining thedifferences between the storage cell, the storage compart-ment and their wild-type counterparts, and on possiblereplacement of cell populations with higher turnover bytheir enzyme supplied precursors. Then, enzyme replace-ment treatment should probably be better conceptualized asan enzyme preventive therapy (see also [19]).

Acknowledgment We wish to thank Transkaryotic Therapies Inc.,Cambridge, MA, USA for providing us with agalsidase alpha andrabbit polyclonal antibody TK88. We also wish to thank the GenzymeCorporation, Cambridge, MA, USA for providing us with agalsidasebeta and the corresponding monoclonal antibodies against α-galactosidase A. We also acknowledge providing samples from patient3 (Dr. G.A. Lammie, Univ. Hospital Wales, Cardiff) and his clinical data(Dr. P. Deegan, Department of Medicine, University of Cambridge).

This work was supported by grants from the Grant Agency ofCharles University, Prague, no. 42/2005/c (to JL), from the GrantAgency of the Czech Republic, no. 303/03H065 (to JK and JL), andby the research project MSM 0021620806 from the Ministry ofEducation and Youth of the Czech Republic (to ME).

Conflict of interest statement We declare that we have no conflictof interest.

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http://rsb.info.nih.gov/ij/

Replacement...AbstractIntroductionPatientsMaterials and methodsExtraction and thin-layer chromatography analysis of (3H)glycosphingolipids

ResultsStudies in cultured mutant cellsUptake of recombinant enzymes into cultured Fabry cells and efficiency of Gb3Cer hydrolysisConfocal microscopic examination of internalized agalsidases with endosomal/lysosomal markers in cultured Fabry cells

Studies in tissuesIn vitro α-GALA activity in the heart samplesImmunohistochemical and ultrastructural studies

DiscussionConclusionsReferences

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